Microbes have developed several different mechanisms of resistance to antibiotics and chemotherapeutic agents. These mechanisms of resistance can be specific, e.g., for a molecule or a family of molecules (e.g., antibiotics), or can be non-specific and be involved in resistance to unrelated antibiotics or other molecules. Several mechanisms of resistance can exist in a single bacterial strain, and those mechanisms may act independently or they may act synergistically to overcome the action of an antibiotic or a combination of antibiotics. Specific mechanisms include degradation of the drug, inactivation of the drug by enzymatic modification, and alteration of the drug target (Spratt (1994) Science 264, 388). There are, however, more general mechanisms of drug resistance, in which access of the antibiotic to the target is prevented or reduced by decreasing the transport of the antibiotic into the cell or by increasing the efflux of the drug from the cell to the outside medium. Both mechanisms can lower the concentration of drug at the target site and allow bacterial survival in the presence of one or more agents which would otherwise inhibit the growth of or kill the bacterial cells.
Efflux pumps are involved in the efflux of drugs from microbial cells. Once in the cytoplasm or periplasm, a drug can be transported back to the outer medium by such membrane-bound protein pumps. Different pumps a specific for a drug or group of drugs, such as the NorA system that transports quinolones, or Tet A that transports tetracyclines, or they can efflux a large variety of molecules, such as certain efflux pumps of Pseudomonas aeruginosa. In general, efflux pumps have a cytoplasmic component and energy is required to transport molecules out of the cell.
Efflux membrane proteins are distributed among microbes, e.g., Gram-positive and Gram-negative bacteria and are involved in transmembrane export of different substances such as heavy metals, organic solvents, dyes, disinfectants and antibiotics (Lawrence, et al. (1998.) Exp. Opin. Invest. Drugs 7, 199-217; Levy (1992) Antimicrob. Agents Chemother. 36, 695-703; Nikaido (1996) J. Bacteriol. 178, 5853; and Sutcliffe (1999) Curr. Opin. Anti-infect. Invest. Drugs 1, 403). Efflux proteins have been broadly classified into five groups: the ATP-binding cassette (ABC) transporters (Fath, et al. (1993) Microbiol. Rev. 57, 995; Ouellette, et al. (1994) Trends in Microbiology 2, 407); RND (Resistance Nodulation and Cell Division, e.g., E. coli AcrB and P. aeruginosa MexB), SMR (Staphylococcal or Small Multidrug Resistance, e.g., S. aureus Smr), MF or MFS (Major Facilitator Superfamily, e.g., all of the Tet efflux proteins, NorA from S. aureus, and Bmr from B. subtilis), and MATE (Multidrug and Toxin Extrusion, e.g., NorM from Vibrio parahaemolyticus).
The exact physiological role of efflux pumps has not yet been clearly defined. Microbial pumps appear to remove intracellular nitrogenous toxins, including polyamines, e.g., spermidine and spermine (Woolridge, et al. (1997) 272, 8864; Woolridge, et al. (1999) Biochem. J. 340, 753), peptides and peptidomimetics (Chen, et al. (2000) Biochem. Biophys. Res. Commun. 269, 743), and antibiotics, e.g., aminoglycosides (Aires, et al. (1999) 43, 2624; Westbrock-Wadman, et al. (1999) 43, 2975; Elkins, et al. (2002) J. Bacteriol. 184(23), 6490; Rosenberg, et al. (2000) J. Bacteriol. 182(6), 1754), allowing cellular survival. They are involved in drug resistance but they also may be involved in the normal physiology of the bacterial cell. For example, the efflux pump coded in the mexA operon of P. aeruginosa has been shown to be regulated by the iron content of the medium, and it is co-regulated with the synthesis of the receptors of siderophores. Siderophores are molecules that are needed for bacterial growth under iron starvation conditions, such as during infection of an animal. They are synthesized in the cytoplasm and exported when the bacterial cell needs iron. Siderophores scavenge iron within the infected animal and return the iron to the microbe to be used for essential microbial processes. Since there is essentially no free iron in the bodies of animals, including the human body, the production of siderophores by infecting bacteria is an important virulence factor for the progress of the infection.
Drug efflux proteins in microbes can mediate resistance causing therapeutic failures. The identification of agents that inhibit the activity of efflux pumps would be of great benefit.